The fibroblast growth factor (FGF) family of signaling polypeptides regulates a diverse array of physiologic functions including mitogenesis, wound healing, cell differentiation and angiogenesis, and development. Both normal and malignant cell growth and proliferation are affected by changes in local concentration of these extracellular signaling molecules, which act as autocrine and paracrine factors. Autocrine FGF signaling may be particularly important in the progression of steroid hormone-dependent cancers to a hormone independent state (Powers et al., Endocr. Relat. Cancer, 2000, 7, 165-197). FGFs and their receptors are expressed at increased levels in several tissues and cell lines, and overexpression is believed to contribute to the malignant phenotype. Furthermore, a number of oncogenes are homologues of genes encoding growth factor receptors, and there is a potential for aberrant activation of FGF-dependent signaling in human pancreatic cancer (Ozawa et al., Teratog. Carcinog. Mutagen., 2001, 21, 27-44).
The two prototypic members are acidic fibroblast growth factor (aFGF or FGF1) and basic fibroblast growth factors (bFGF or FGF2), and to date, at least twenty distinct FGF family members have been identified. The cellular response to FGFs is transmitted via four types of high affinity transmembrane tyrosine-kinase fibroblast growth factor receptors numbered 1 to 4 (FGFR-1 to FGFR-4). Upon ligand binding, the receptors dimerize and auto- or trans-phosphorylate specific cytoplasmic tyrosine residues to transmit an intracellular signal that ultimately reaches nuclear transcription factor effectors. Mitogenic signaling by these FGFRs is subsequently mediated via a number of pathways, including the ras/raf/MAP kinase cascade (Ozawa et al., Teratog. Carcinog. Mutagen., 2001, 21, 27-44).
Alternative splicing of the mRNA from the FGFRs 1, 2, and 3 results in a wide range of receptor isoforms with varying ligand-binding properties and specificities. With seven different receptor possibilities and at least 20 ligands in the FGF family, there is a great deal of diversity in the FGF signaling pathway (Powers et al., Endocr. Relat. Cancer, 2000, 7, 165-197). Furthermore, expression and localization of the receptor isoforms is regulated in a tissue specific manner. Thus, the various FGFs may exert different influences upon different cell types by interacting with different receptor splice variants to initiate unique intracellular signaling cascades, leading to a panoply of cellular responses (Ozawa et al., Teratog. Carcinog. Mutagen., 2001, 21, 27-44).
Fibroblast growth factor receptor 2 (also known as FGF receptor-2, FGFR-2, Crouzon syndrome, craniofacial dysostosis 1 (CFD1), Pfeiffer syndrome, JWS, CEK3, ECT1, TK14, TK25, BFR-1, and K-Sam) as well as its splice variant, keratinocyte growth factor receptor (KGFR or bek), have high affinity for the acidic and/or basic fibroblast growth factors, as well as the keratinocyte growth factor ligands.
The human fibroblast growth factor receptor 2 gene was originally isolated as the protein tyrosine kinase 14 (TK14) from a human tumor cDNA library. High levels of amino acid homology to the chicken bFGF receptor, to a partial sequence of the mouse bek protein, and to another human protein encoded by the fms-like tyrosine kinase gene were noted, indicating that the human genome had at least two distinct fibroblast growth factor receptors. As further evidence that this was a true cell-surface receptor, overexpression of the TK14 gene in COS-1 cells lead to the appearance of new cell-surface binding sites for acidic and basic FGFs (Houssaint et al., Proc. Natl. Acad. Sci. U.S.A., 1990, 87, 8180-8184). Independently, fibroblast growth factor receptor 2 was cloned from a human brainstem cDNA library, using the murine bek gene as a probe (Dionne et al., Embo J., 1990, 9, 2685-2692). The human fibroblast growth factor receptor 2 gene was mapped to the 10q25.3-q26 locus (Dionne et al., Cytogenet. Cell. Genet., 1992, 60, 34-36).
The keratinocyte growth factor receptor, a splice variant related to, but distinct from, fibroblast growth factor receptor 2, was cloned from a B5/589 mammary epithelial cell cDNA library (Miki et al., Science, 1991, 251, 72-75). KGF is expressed only by mesenchymal cells, such as fibroblasts and hair follicular dermal papilla cells, and KGFR is expressed only by epithelial tissues, such as epidermis and intestinal epithelium, generally in close proximity to cells that express KGF. This splice variant of fibroblast growth factor 2 is an important mediator of proliferation and differentiation in a wide variety of epithelial cells including hepatocytes, gastrointestinal epithelial cells, type II pneumocytes, transitional urothelial cells, and keratinocytes in all stratified squamous epithelia (Danilenko, Toxicol. Pathol., 1999, 27, 64-71).
Fibroblast growth factor receptor 2 also propagates the potent osteogenic effects of FGFs during osteoblast growth and differentiation. Mutations in fibroblast growth factor receptor 2, leading to complex functional alterations, were shown to induce abnormal ossification of cranial sutures (craniosynostosis), implying a major role of FGFR signaling in intramembranous bone formation. For example, in Apert (AP) syndrome, characterized by premature cranial suture ossification, most cases are associated with point mutations engendering gain-of-function in fibroblast growth factor receptor 2 (Lemonnier et al., J. Bone Miner. Res., 2001, 16, 832-845).
Several severe abnormalities in human skeletal development, including Apert, Crouzon, Jackson-Weiss, Beare-Stevenson cutis gyrata, and Pfeiffer syndromes are associated with the occurrence of mutations in fibroblast growth factor receptor 2. Most, if not all, cases of Pfeiffer Syndrome (PS) are also caused by de novo mutation of the fibroblast growth factor receptor 2 gene (Meyers et al., Am. J. Hum. Genet., 1996, 58, 491-498; Plomp et al., Am. J. Med. Genet., 1998, 75, 245-251), and it was recently shown that mutations in fibroblast growth factor receptor 2 break one of the cardinal rules governing ligand specificity. Namely, two mutant splice forms of fibroblast growth factor receptor, FGFR2c and FGFR2b, have acquired the ability to bind to and be activated by a typical FGF ligands. This loss of ligand specificity leads to aberrant signaling and suggests that the severe phenotypes of these disease syndromes result from ectopic ligand-dependent activation of fibroblast growth factor receptor 2 (Yu et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 14536-14541).
In keeping with the findings in humans, the heterozygous abrogation of one fibroblast growth factor receptor 2 allele in mice resulted in a splicing switch leading to a gain-of-function mutation. The consequences of this mutation are neonatal growth retardation and death, ocular proptosis, precocious ossification of the coronal sutures, zygomatic arch joints, and sternabrae, and abnormalities in secondary branching of organs that undergo branching morphogenesis in development, as well as other major defects in the kidney, lung, and lacrimal glands. This phenotype strongly parallels those of Apert and Pfeiffer syndrome patients (Hajihosseini et al., Proc. Natl. Acad. Sci. U.S.A., 2001, 98, 3855-3860).
Gene rearrangements of fibroblast growth factor receptor 2 have also been shown to lead to ligand independent activation of FGFRs (Powers et al., Endocr. Relat. Cancer, 2000, 7, 165-197). A constitutively active form of fibroblast growth factor receptor 2 with an altered C-terminus was identified in a rat osteosarcoma cDNA library, and this isoform arose from a chromosomal rearrangement of the fibroblast growth factor receptor 2 gene with a novel gene, FGFR activating gene 1 (FRAG1). The FGFR-2/FRAG1 fusion protein seems to form constitutive dimers, resulting in autophosphorylation of the fibroblast growth factor receptor 2 kinase domains and activation of the FGF signaling pathway (Lorenzi et al., Proc. Natl. Acad. Sci. U.S.A., 1996, 93, 8956-8961).
KGFR and fibroblast growth factor receptor 2 mRNAs were found to be overexpressed in both human pancreatic cancer cells and the adjacent pancreatic parenchyma (Ishiwata et al., Am. J. Pathol., 1998, 153, 213-222), and expression of the FGFR2-IIIb isoform of fibroblast growth factor receptor 2 is downregulated in a subset of transitional cell carcinomas of the bladder, further indicating the involvement of this gene in tumorogenesis (Ricol et al., Oncogene, 1999, 18, 7234-7243).
The modulation of fibroblast growth factor receptor 2 activity and/or expression is an ideal target for therapeutic intervention aimed at regulating the FGF signaling pathway in the prevention and treatment of many cancers and hyperproliferative diseases.
Investigative strategies aimed at modulating fibroblast growth factor receptor 2 function have involved the use of antibodies directed against a peptide fragment of fibroblast growth factor receptor 2 to perturb ligand-receptor binding and functionally block signaling, and the use of antisense oligonucleotides.
A phosphorothioate antisense oligodeoxynucleotide 15 nucleotides in length which does not discriminate between any of the alternate splice forms of fibroblast growth factor receptor 2, spanning the translation start site was used to investigate the role of fibroblast growth factor receptor 2 in FGF signaling. In the same study, two additional phosphorothioate antisense oligodeoxynucleotides 19 and 16 nucleotides in length were designed to be specific for KGFR and bek exon sequences, respectively (Post et al., Development, 1996, 122, 3107-3115). These antisense oligonucleotides were used to show that lung branching morphogenesis is reduced in cultures of embryonic rat lung explants when expression of fibroblast growth factor receptor 2 is abrogated. KGFR-specific antisense oligonucleotides dramatically inhibited lung branching, while bek-specific antisense oligonucleotides reduced lung branching to a lesser degree (Post et al., Development, 1996, 122, 3107-3115).
A phosphorothioate antisense oligodeoxynucleotide of unspecified length, complementary to the translation start site of fibroblast growth factor receptor 2 was used to investigate the role of fibroblast growth factor receptor 2 signaling in human glioblastoma cells (Yamada et al., Glia, 1999, 28, 66-76).
Disclosed and claimed in U.S. Pat. No. 5,578,566 are KGFR peptides which inhibit binding between keratinocyte growth factor (KGF) and the FGFR-1, FGFR-2 and KGFR receptors. The sequence of the peptides is derived from regions in the receptors which specifically bind the growth factor. Also provided are pharmaceutical compositions and methods of inhibiting the interaction of KGF and the receptor in a patient to treat various carcinomas (Bottaro et al., 1996).
Disclosed and claimed in U.S. Pat. No. 6,054,312 is a composition comprising a physiologically acceptable buffer and filamentous phage particles presenting a ligand that binds to a mammalian cell surface molecule, wherein the cell surface molecule is an FGF receptor, and the phage genome encodes a therapeutic gene product which is an antisense oligonucleotide (Larocca et al., 2000).
Currently, there are no known therapeutic agents that effectively inhibit the synthesis of fibroblast growth factor receptor 2. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting fibroblast growth factor receptor 2 function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and therefore may prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of fibroblast growth factor receptor 2 expression.
The present invention provides compositions and methods for modulating fibroblast growth factor receptor 2 expression, including modulation of the truncated mutants and alternatively spliced forms of fibroblast growth factor receptor 2 such as KGFR.